The kinetic mechanism of cyclic adenosine 3 feet, 5 feet monophosphate dependent protein kinase from bovine skeletal muscle will be determined in two parts: the order of addition of substrates and release of products and the location of slow steps along the reaction pathway. Techniques are available to obtain this information which include initial rate studies in the presence and absence of products and dead-end inhibitors, isotope exchange at equilibrium and isotope trapping. Advances in steady state kinetic theory have shown that the chemical catalytic mechanism can also be obtained using a determination of the pH variation of kinetic parameters (for example Vmax, V/K, and Ki for a substrate, inhibitor or activator), and chemical modification using specific functional reagents. It is the goal of this project to determine the mechanism of action of the protein kinase catalytic subunit. Since this enzyme is intimately involved in cellular regulation, a detailed study of its mechanism is particularly appropriate. This kind of approach has been used with success for several enzymes including hexokinase, fructokinase, creatine kinase, and aspartic aminotransferase, but only a very small number of enzymes have mechanisms which are known with any degree of certainty.